Quantifying the antimicrobial activity of CRISPR-Cas9-accA modified Δ B. subtilis mutants against V. harveyi and E. Coli

  1. Tatiana Hillman

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Abstract

Probiotics are increasingly popular, currently. Probiotics have been described with the ability to treat many disorders of the gastrointestinal tract (GIT) such as irritable bowel syndrome (IBS) and Crohn’s disease. Types of probiotics include bacterial strains from Lactobacillus and Bifidobacterium . Probiotics can restore balance to gut microbiota by outcompeting pathogenic bacteria for nutrients and secrete antimicrobials to eliminate these bacterial pathogens. However, the viability of most advertised probiotics lose their potency due to being freeze dried into powders during storage or for consuming. Many probiotics become ineffective and produce lower CFUs while traversing through the gastric acids of the digestive system. For these reasons, this study sought to enhance the antimicrobial response of a highly potent probiotic known as Bacillus subtilis. B. subtilis has been used to treat many disorders of the gut and secrete many antimicrobials lethal for pathogenic microbes. B. subtilis was genetically modified to express CRISPR-Cas9 nuclease deletion of the accA gene (Δ B.subtilis mutants), which inhibits expression of an essential accA gene a part of the fatty acid synthesis (FAS) metabolic pathway. The CRISPR-Cas9- accA ΔB.subtilis mutants were co-cultured with V. harveyi and E. Coli . Bacterial growth, biofilm formation, antimicrobial activity, and antibiotic resistance were quantified. It was found that ΔB.subtilis mutants co-cultured with V. harveyi and E. Coli lessened bacterial growth, amplified biofilm with V. harveyi , reduced biofilm formation of E. Coli , the co-cultures with the mutants lacked antimicrobial activity, and increased the antibiotic resistance of V. harveyi and E. Coli . It can be concluded that there is an immense potential for using genetically engineered probiotic strains to enhance the antimicrobial activity of B. subtilis , which can amplify the reduction of pathogenic bacteria. However, the safety and frugality of using B. subtilis as a probiotic requires further consideration.

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  1. PREreview

    This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/7240736.

    This review was written by an undergraduate at Mount Holyoke College (MA, USA) who selected this preprint for an assignment in a course on peer review taught by Dr. Rebeccah S. Lijek, Assistant Professor of Biological Sciences.

    Disclosures: The review author declares no conflict of interest and has no personal or financial relationship with the study's author. The reviewer acknowledges a limitation of this review due to the narrow scope of their expertise as an undergraduate student.

    Summary:

    The preprint started with introducing Bacillus subtilis as an effective antimicrobial probiotic to balance the microflora in the gastrointestinal tract. Though the specific mechanism is unclear, it seems that B. subtilis can affect cell growth and the potency of other gut microbiota. Thus, it is worthy to study the probiotic antimicrobial mechanism of B. subtilis and investigate whether this potentiality changes in its mutant. The goal of the study was examine the potency of genetically engineered probiotic strains to decrease the pathogenic bacteria and avoid dysbiosis in the human gut. The study researched the B. subtilis probiotic antimicrobial effect by using mutant B. subtilis, which is synthetic and genetic reengineering of the wild-type B. subtilis by the method of the CRISPR-Cas9 plasmid vectors. The bacterial growth, biofilm formation, antimicrobial activity, and antibiotic resistance were quantified in the experiment. The result showed lessening bacterial growth in mutant B.subtilis co-cultured with V. harveyi and E. Coli, decreasing biofilm formation with E. Coli and increasing biofilm formation with V. harveyi, increasing antibiotic resistance of V. harveyi and E.Coli, and no antimicrobial activity observed since the co-cultures are non-pathogenic.

    Comments:

    The background information about the probiotics and their relation to the gastrointestinal tract offered in this preprint is sufficient to understand the importance of the research question, and the study referred to relevant literature to facilitate it. The overall study design was appropriate, and each section of the method was well described and designed for the research question. The results were generally clearly presented by the figures or charts and explained in the legends and the paragraphs except for the minor flaws. 

    However, the main problem in this preprint is that the rationale for the study is ambiguous. The mechanism of how CRISPR-mutated B.subtilis affects the growth of pathogenic bacteria doesn't elaborate on in the introduction. Moreover, the conclusion that genetically engineered probiotics enhance the antimicrobial activity of B. subtilis was not firmly strengthened in this experiment due to the lack of antimicrobial activity in the non-pathogenic co-cultures. To further address the research question, there is a great need to run the experiment with pathogenic microbes to better observe the antimicrobial activity. The author also acknowledged this limitation in the discussion, indicating that the study did not detect the antimicrobial activity of the mutant B. subtilis in co-cultures because V. harveyi and E. Coli bacterial strains are non-pathogenic.

    The study is novel and has a potential impact on the field. Considering the decreasing effect of advertised probiotics during the transition to the GI tract, the potential enhancement of antimicrobial effectiveness of mutant B.subtilis can be applied to treat disease or balance microflora in the gut. Yet, the author also mentioned that the application of probiotics to consumers needs cautiousness because the probiotics share in transferring antibiotic-resistant genes between pathogenic and commensal bacteria. There are also some unsolved problems and challenges left over. For example, the mechanisms of probiotic properties are still not understood comprehensively. In addition, future research needs to investigate and distinguish the most effective probiotics with minimal toxins. They also need to find a frugal and practical drug delivery method to deliver probiotics into the target region without losing their potency. 

    Strengths:

    1. The preprint provides sufficient background information on probiotics, including their relationship to the gastrointestinal tract, which makes this paper more accessible to the general audience.
    2. The structure of the methods and materials section is organized and concise. The procedure is also detailed enough for further replication of the same experiment.

     

    Major issues:

    1. The justification for the hypothesis or the purpose of the study is unclear and insufficient. Even though it explains the deletion of the accA gene inhibits fatty acid formation thereby decreasing the growth of bacteria, the mechanism of how accA deleted B.subtilis reduce pathogenic bacteria through co-culture doesn't mention in the introduction. It should include why changing B. subtilis itself can influence other bacteria in the co-cultures, and how they interact during this process. 
    2. Why specifically choose to delete the accA subunit instead of other subunits of acetyl-CoA carboxyl transferase enzymes such as accB, accC, and accD? It should include the reason of a brief explanation in the introduction part.
    3. In the introduction part, it introduces that the Vibrio harveyi is "a highly pathogenic bacteria found in many of the fish sources available for human consumption." However, in the "antimicrobial activity" session under the discussion part, the last several sentences are opposite to the one above. For example, the sentences "No antimicrobial activity was detected because the V. harveyi and E. Coli bacterial strains co-cultured with the mutant B. subtilis, were non-pathogenic" and "Therefore, the findings of antimicrobial activity via the cross-streak method, in this study, was limited due to lack of access to pathogenic target bacterial strains" illustrate V.harveyi are non-pathogenic. 
    4. In the "co-culture assays" part, it is confusing whether you meant the combination of V. harveyi, E. Coli, and mutant B. subtilis these all together or it is V. harveyi and mutant B. subtilis as a combination, and E.Coli and mutant B. subtilis as another combination. Using more clear sentences to illustrate it would help.
    5. There are some repetitions between the result and discussion sections. I suggest paring down the result section by only including the representative data concisely without further interpretation.
    6. The antimicrobial activity was not observed in this experiment which concludes the antimicrobial activity of mutant B. subtilis can be enhanced untenable. Further research is required to have the conclusion, but at this point, I suggest changing the way to phrase the conclusion would be more precise.

     

    Minor issues:

    General: 

    1. There are some typos (e.g. misspellings or missing periods) throughout the paper, which might affect the understanding especially when there is a lot of jargon. For example, in figure 4, according to the legend, there is a typo in the variable name in graph A. It should be "WT B.sub& V.H." instead of "WT B.sub".

    Methods and Materials:

    1. In "B.subtilis Transformation", when transforming the plasmid into the B.subtilis, what induces the CRISPR to work, is it the 2% xylose? It is important to mention the mechanism.
    2. In "Co-culture Assays", what I understand about the combination of colonies for one group is consisting of 5-6 colonies each from E.Coli BL21, E.Coli DH5a, V. harveyi, and mutant B. subtilis. I am curious why having two different E. Coli types here, so mentioning it in the experiment design will make more sense.

    Results:

    1. In the co-culture assays part, the paragraph claims the result of decreased bacterial growth in V. Harveyi, E. Coli, and ΔB.subtilis mutant, but the figure only includes the data of co-culture between V. Harveyi and ΔB.subtilis mutant. The method section of the co-culture assay also mentions the setup of the controlled group which is the co-culture among wild-type B.subtilis, V. Harveyi, and E. Coli, but the result part doesn't mention it. I suggest including the data of co-culture between E. Coli and ΔB.subtilis mutant as well as the co-culture of the control group to keep the consistency.
    2. In the figure 6 legend, how does the conclusion "ΔB.subtilis mutant-V.H and ΔB.subtilis mutant-E. Coli co-cultures showed more antibiotic resistance than the control samples" come out? It is confusing.
    3. The variable names in figure 7 and figure 8 are confusing and unclear. For example, "WT" stands for which bacteria's wild type? Or does it mean the co-culture of V. Harveyi. and wild-type B.subtilis? The word usage should be more careful.
  2. Version published to 10.1101/2021.08.10.455802v1 on bioRxiv